The trypsin sub-family of serine proteases (referred to as the trypsin-like serine proteases) is composed of proteases which hydrolyze peptide bonds that follow an arginine or lysine residue. These proteases play an important physiological role in digestion, coagulation, fibrinolysis, blood pressure regulation, fertility, and inflammation. "Design of Enzyme Inhibitors as Drugs", Oxford Science Publications,(Edits. Sandler, M., Smith, H. J. 1989). Selective inhibitors of trypsin-like serine proteases are thought to be useful as drugs for intervention into many disease states in which the involvement of these proteases has been implicated.
Peptide analogs which utilize the catalytic mechanism of an enzyme (e.g. transition-state inhibitors) have been suggested as inhibitors of the trypsin-like serine proteases. The catalytic mechanism of these proteinases is thought to involve the attack of the active-site serine on the carbonyl bearing the scissile amide bond of the substrate, to give a tetrahedral intermediate which subsequently results in peptide bond cleave. It has been reported that peptide analogs which are stable mimics of this tetrahedral intermediate (i.e., transition-state analogs) can be selective enzyme inhibitors. Delbaere, L. T. J., Brayer, G. D., J. Mol. Biol. 183:89-103, 1985 and "Proteases and Biological Control", Cold Spring Harbor Laboratory Press, pp. 429-454 (Edits. Aoyagi, T. and Umezawa, H. 1975). Selective transition-state inhibitors of the trypsin-like serine proteases may therefore be useful as drugs for intervention into many disease states in which the involvement of these proteases has been implicated.
One candidate group of transition-state inhibitors which may be particularly useful are the peptide analogs which have an aldehyde group on the C-terminus of the peptide analog. Peptide aldehydes were initially discovered as natural products produced by a number of actinomycete strains. Some derivatives of natural products have been reported to be selective inhibitors of various types of serine and cysteine proteinases. Aoyagi, T., Supra. For example, the peptide, alaninal elastatinal, was reported to be a potent elastase inhibitor, but not an inhibitor of trypsin or the trypsin-like serine proteases. Hassall, C. H. et al., FEBS Lett., 183:201-205 (1985). Elastase inhibitors are of interest in the treatment of diseases such as emphysema and synthetic peptide aldehydes have been reported to be excellent inhibitors of human leukocyte elastase. Sandler, M., Smith, H. J., Supra. It has been reported that the selectivity of these naturally occurring analogs has been enhanced by modifying the peptide sequence. Bajusz, S. et al., J. Med. Chem. 33:1729-1735 (1990); and McConnell, R. M. et al., J. Med. Chem. 33: 86-93 (1990).
The peptidyl argininal, leupeptin (Acetyl-L-Leu-L-Leu-L-Arg-al), has been reported to be a selective inhibitor of trypsin-like serine proteases. "Structures and activities of protease inhibitors of microbial origin", Proteases and Biological Control, Cold Spring Harbor Laboratory Press, pp. 429-454 (Edits. Aoyagi, T., Umezawa, H. 1975). Leupeptin, along with its naturally occurring variants and synthetic analogs, have been reported to be potent inhibitors of several trypsin-like serine proteases in the coagulation cascade.
The peptide argininal, D-Phe-L-Pro-L-Arg-al, and analogs thereof, have been reported to show a marked selectivity for particular coagulation factors. For example, one such analog (N-methyl-D-Phe-Pro-Arg-al) has been developed as a thrombin inhibitor and is reported to have significant in vivo anticoagulant activity. U.S. Pat. Nos. 4,316,889 (1982), 4,399,065 (1983), 4,478,745 (1984), 4,346,078 (1982), and 4,708,039 (1987).
A major problem in medical research directed to the use of peptidyl aldehydes as potential drugs for intervention into many disease states in which trypsin-like serine proteases have been implicated has been the difficulty in synthesizing the peptidyl argininals. Though solution-phase methods for their synthesis have been reported, their synthesis remains a labor-intensive and time-consuming process.
Three methods for the solution-phase synthesis of peptidyl argininals (Arg-al), each using a different intermediate, have been reported.
The use of L-Leu-L-Arg-al dibutylacetal as an intermediate has been reported in the synthesis of more than 30 peptidyl argininals. In particular, L-Leu-L-Arg-al was reported to be prepared by thermolysin digestion of leupeptin (acetyl-L-Leu-L-Leu-L-Arg-al), transformation of the digestion product to a racemic dibutyl acetal (L-Leu-D,L-Arg-dibutylacetal), followed by separation of the diastereomers. Saino, T et al., Chem. Pharm. Bull., 30(7):2319 (1982); T. Saino et al., J. Antibiotics, 41:220 (1988).
The use of the N.sup..omega. -carbobenzyloxy-arginine lactam as an intermediate in the synthesis of peptidyl argininals has been reported. The lactam was reported to be coupled to a variety of peptides in good to high yield. The resulting peptidyl-N.sup..omega. -carbobenzyloxy-arginine lactam was reduced with LiAlH.sub.4 to form the peptidyl-N.sup..omega. -carbobenzyloxy-argininal, and subsequently hydrogenated to give the peptidyl argininal. Basjusz, S. et al, J. Med. Chem., 53: 1729 (1990); Shuman, R. T. et al., J. Med. Chem., 36:314 (1993); Balasubramanian, N. et al., J. Med. Chem., 36:300 (1993).
The use of semicarbazone intermediates has been reported in the synthesis of peptidyl argininals. The unsubstituted semicarbazone, N.sup.g -nitro-L-argininal semicarbazone, was used as an intermediate in the synthesis of peptidyl argininals. McConnell, R. M. et al., J. Med Chem., 33:86 (1990); R. M. McConnell, J. L. York, D. Frizzell, C, Ezell, J. Med Chem., 36, 1084-1089 (1993). N.sup.g -nitro-L-argininal semicarbazonyl-4-methylcyclohexane carboxylic acid was reported as an intermediate in the preparation of peptide aldehydes by a solid phase method. Murphy, A. M. et al., J. Am. Chem. Soc., 114:3156 (1992); and Webb, T. R., U.S. Pat. No. 5,283,293 (Feb. 1, 1994). N.sup.g -nitro-L-argininal semicarbazonyl-4-diphenylmethane was reported as an intermediate for the solution-phase synthesis of peptidyl argininals. Brunck, T. K. et al., WO 93/14779 (1993).